The coagulation of blood involves a cascading series of reactions leading to the formation of fibrin. The coagulation cascade consists of two overlapping pathways required for hemostasis. The intrinsic pathway comprises protein factors present in circulating blood, while the extrinsic pathway requires tissue factor which is expressed on the cell surface of a variety of tissues in response to vascular injury. Agents interfering with the coagulation cascade, such as heparin and coumarin derivatives, have well known therapeutic uses in the prophylaxis of venous thrombosis.
Aspirin also provides a protective effect against thrombosis. It induces a long-lasting functional defect in platelets, detectable clinically as a prolongation of the bleeding time, through inhibition of the cyclooxygenase activity of the human platelet enzyme prostaglandin H-synthase (PGHS-1) with doses as low as 30 to 75 mg. Since gastrointestinal side effects of aspirin appear to be dose-dependent, and for secondary prevention, treatment with aspirin is recommended for an indefinite period, there are practical reasons to choose the lowest effective dose. Further it has been speculated that a low dose (30 mg daily) might be more antithrombotic but attempts to identify the optimal dosage have yielded conflicting results. It has been claimed that the dose of aspirin needed to suppress fully platelet aggregation may be higher in patients with cerebrovascular disease than in healthy subjects and may vary from time to time in the same patient. However, aspirin in any daily dose of 30 mg or higher reduces the risk of major vascular events by 20% at most, which leaves much room for improvement. Further, the inhibiting role of aspirin may lead to prevention of thrombosis and to excess bleeding. The balance between the two depends critically on the absolute thrombotic versus hemorrhage risk of the patient.
In patients with acute myocardial infarction, reduction of infarct size, preservation of ventricular function and reduction in mortality has been demonstrated with various thrombolytic agents. However these agents still have significant shortcomings, including the need for large therapeutic doses, limited fibrin specificity, and significant associated bleeding tendency. Recombinant tissue plasminogen activator (t-PA) restores complete patency in just over one half of patients, whereas streptokinase achieves this goal in less than one third. Further, reocclusion after thrombolytic therapy occurs in 5 to 10% of cases during the hospital stay and in up to 30% within the first year according to Verheugt et al., J. Am. Coll. Cardiol. (1996) 27:618-627. Numerous studies have examined the effects of adjunctive antithrombin therapy for patients with acute myocardial infarction. For instance, U.S. Pat. No. 5,589,173 discloses a method for dissolving and preventing reformation of an occluding thrombus comprising administering a tissue factor protein antagonist, such as a monoclonal or polyclonal antibody, in adjunction to a thrombolytic agent.
In arterial blood flow, the platelet adhesion is mainly supported by the platelet receptor glycoprotein (GP) lb which interacts with von Willebrand factor (vWF) at the site of vessel wall injury. Blood platelets, through the processes of adhesion, activation, shape change, release reaction and aggregation, forrn the first line of defence when blood vessels are damaged. They form a hemostatic plug at the site of injury to prevent excessive blood loss. Extensive platelet activation however may overcome the normal thrombo-regulatory mechanisms that limit the size of the hemostatic plug. Platelets then become major prothrombotic offenders predisposing to vaso-occlusive disease.
The formation of a platelet plug during primary haemostasis and of an occluding thrombus in arterial thrombosis involves common pathways. The first event is platelet adhesion to subendothelial collagen, exposed upon vessel injury, which can be a ruptured atherosclerotic plaque. Circulating vWF binds to the collagen and, under the influence of high shear stress, undergoes a conformational change allowing it to bind to its receptor, GPIb/IX/V, on the platelet membrane. This interaction is essential in order to produce a thrombus, at least in smaller vessels or stenosed arteries where shear stress is high, and results in slowing down the progress of the platelets across the damaged surface. Full immobilisation of platelets occurs when collagen binds to its receptor GPIa/IIa (integrin α2β1). In addition, collagen activates platelets mainly by binding to GPVI, another collagen receptor. When platelets are activated, GPIIb/IIIa (integrin αIIBβ3) undergoes a conformational change and acquires the ability to bind to fibrinogen and vWF which crosslink adjacent platelets to finally form platelet aggregates.
Lately much effort has been directed to develop antibodies and peptides that can block the binding of the adhesive proteins to GPIIb/IIIa and many of these are being tested in clinical trials. One approach to blocking platelet aggregation involves monoclonal antibodies specific for GPIIb/IIIa receptors. Specifically, a murine monoclonal antibody named 7 E3 useful in the treatment of human thrombotic diseases is described in EP-A-206,532 and U.S. Pat. No. 5,387,413. However it is known in the art that murine antibodies have characteristics which may severely limit their use in human therapy. As foreign proteins, they may elicit an anti-immunoglobulin response termed human anti-mouse antibody (HAMA) that reduces or destroys their therapeutic efficacy and/or provokes allergic or hypersensitivity reactions in patients, as taught by Jaffers et al., Transplantation (1986) 41:572. The need for re-administration in therapies of thromboembolic disorders increases the likelihood of such immune reactions. While the use of human monoclonal antibodies would address this limitation, it has proven difficult to generate large amounts of such antibodies by conventional hybridoma technology.
Recombinant technology has therefore been used to construct “humanized” antibodies that maintain the high binding affinity of murine monoclonal antibodies but exhibit reduced immunogenicity in humans. In particular, there have been suggested chimeric antibodies in which the variable region (V) of a non-human antibody is combined with the constant (C) region of a human antibody. As an example, the murine Fc fragment was removed from 7E3 and replaced by the human constant immunoglobulin G region to form a chimera known as c7E3 Fab or abciximab. Obtention of such chimeric immunoglobulins is described in detail in U.S. Pat. No. 5,770,198.
The potential for synergism between GPIIb/IIIa inhibition by monoclonal antibody 7E3 Fab and thrombolytic therapy was evaluated by Kleiman et al., J. Am. Coll. Cardiol (1993) 22:381-389. Major bleeding was frequent in this study. Hence, the potential for life-threatening bleeding is clearly a major concern with this combination of powerful anti-thrombotic compounds.
The GPIb-vWF axis therefore presents an attractive alternative to GPIIb/IIIa-fibrinogen as a target for platelet inhibition, since a suitable inhibitor might be expected to down regulate other manifestations of platelet activity such as granule release, thought to play a role in the development of arteriosclerosis. Activation of platelets is accompanied by secretion of vasoactive substances (thromboxane A2, serotonin) as well as growth factors such as PGDF. Therefore, early inhibition of platelet activation and hence prevention of the secretion of their growth and migration factors, via a GPIb blocker, would reduce the proliferation of smooth muscle cells and restenosis after thrombolytic therapy. Moreover, the interaction of GPIb with the damaged vessel wall (adhesion, as well as aggregation and secretion of platelet content) is highly blood flow dependent. Unlike GPIIb/IIIa interactions, GPIb-vWF interaction occurs exclusively under the high flow conditions, as occurs in small arteries or created by arterial stenoses. Hence, GPIb inhibition represents theoretically an ideal way to target effective platelet inhibition to damaged arterial areas. GPIb inhibition therefore appears particularly suited to treat patients with acute coronary syndromes, transient cerebral attacks and claudication due to peripheral arterial diseases, including prevention of the frequently letal thrombotic complications of acute coronary syndromes, angioplasty, unstable angina and myocardial infarction.
Despite these potential advantages, the development of compounds that interfere with the vWF-GPIb axis has lagged behind. Only a few in vivo studies described the effects of inhibition of platelet adhesion on thrombogenesis. They include the use of anti-vWF monodonal antibodies, GPIb binding snake venom proteins like echicetin and crotalin, aurin tricarboxylic acid that binds to vWF and recombinant vWF fragments like VCL, all of which inhibit vWF-GPIb interaction. All these molecules were anti-thrombotic, particularly in studies where a thrombus was formed under high shear conditions. U.S. Pat. No. 5,486,361 discloses a monoclonal antibody 4H12 which specifically binds to the α chain of GPIb and, by means of this interaction, totally inhibits the binding of thrombin to normal human platelets. In addition, it inhibits more than 90% of thrombin-induced von Willebrand factor or fibrinogen binding to platelets. Further, 4H12 does not inhibit ristocetin- or botrocetin-induced binding of von Willebrand factor to platelet cells, which indicates that this antibody does not prevent von Willebrand factor binding to GPIb. A number of potent inhibitory anti-GPIb antibodies, such as LJIb1 disclosed by F. Pareti et al. in British Journal of Haematology (1992) 82, 81-86, have been produced and were extensively tested with respect to their in vitro effect under both static (platelet agglutination, vWF-binding) and flow conditions. However for none of these anti-human GPIb antibodies an in vivo anti-thrombotic effect could be demonstrated. In vivo data obtained by B. Becker and J. L. Miller (Blood (1989)2:680-694) describe the effect of injecting guinea pigs with intact antibody or F(ab′)2 fragments of PG1, a monoclonal anti-guinea pig GPIb antibody. After intraperitoneal injection of the intact antibody, a hemorrhagic state was produced with a significant lengthening of the bleeding time and drop of the platelet count to 50% of its baseline value. Injection of 0.53 to 2.5 mg/kg of the F(ab′)2 fragments did not decrease the platelet count more than 21%, and bleeding times never increased by more than one minute over baseline values. However, in this particular study the antithrombotic effect of the F(ab′)2 fragments was not further investigated by e.g. testing the fragments in an animal thrombosis model. In a follow-up study J. L. Miller et al., Arterioscier. Thromb. (1991) 11:1231-6 disclosed that the F(ab′)2 fragments of PG1 in guinea pigs using these could effectively reduce thrombus formation on a laser-induced injury. Unfortunately, this antibody does not cmss react with human platelets and therefore it has no further clinical relevance for human therapy.
Part of this rather surprising lack of in vivo studies is due to the low cross reactivity of the anti-human GPIb monoclonal antibodies with platelets from commonly used laboratory animals. This predisposes to the use of non-human primates as experimental animals. However, even then attempts to perform in vivo studies are hampered because injection of the anti-GPIb monoclonal antibodies, as well as the snake venom protein echicetin that reacts with GPIb, invariably causes severe thrombocytopenia, as taught by US-A-5,336,667. WO-A-002667 further discloses monoclonal antibodies Fab fragments but does not discuss thrombocytopenia and does not mention in vivo tests.
One persistent concern with all available thrombolytic and anti-thrombotc agents, including aspirin, is to induce a risk of overdose and therefore of excessive and life-threatening bleeding. Therefore a first goal of the present invention is to provide a thrombus formation protective means by providing a platelet adhesion inhibitor that does not induce a risk of bleeding. A second goal of the present invention is to provide a thrombus formation protective means by providing an inhibitor of platelet adhesion without incurring the risk of thrombocytopenia, A third goal of the present invention is the targetting of platelet adhesion, activation and aggregation under high shear conditions, which is of particular importance in the setting of highly stenotic atherosclerotic lesions. The specific targetting of highly stenotic areas in the circulation should make GPIb inhibition particularly suitable for treating unstable angina and in the chronic prevention of arterial occlusion. Unlike with GPIIb/IIIa inhibition, platelet aggregation as well as hemostasis is not expected to be inhibited in low shear vessels, i.e. in veins and normal arteries. Bleeding complications from these vessels by inhibition of GPIb may therefore be expected to be better reduced than with GPIIb/IIIa inhibition.